Interferon-induced IFITM recruitment of ZMPSTE24 blocks viral endocytic entry Abstract Viruses must penetrate the cell?s protective phospholipid bilayer to initiate the infectious process. Enveloped viruses enter the cytoplasm by a membrane fusion mechanism often via an endolysosomal route. Interferon- inducible transmembrane proteins (IFITM) were shown to block a broad spectrum of RNA viruses with endocytic entry. However, how IFITM proteins inhibit viral entry is not clear. This application proposes that ZMPSTE24 (abbreviated ZMP), an evolutionarily conserved membrane associated zinc metalloprotease, is a broad-spectrum antiviral effector downstream of IFITM. Preliminary data suggest that type I interferon (IFN) induces IFITM proteins, which in turn recruit and retain ZMP on endosomes. Experiments involving overexpression and genetic deficiency indicate ZMP has antiviral activity against RNA and DNA viruses, including influenza A virus (IAV), vesicular stomatitis virus (VSV) and vaccinia virus (VACV). Direct viral entry assays using virus-like particles suggest ZMP restricts viral endosomal entry. To understand how ZMP functions, this proposal probes the molecular mechanism controlling ZMP cooperation with IFITM proteins. Aim 1 will determine how ZMP and IFITM cooperate and if either can function independently to restrict viral infection. Critical residues involved in ZMP-IFITM interaction and antiviral function are identified. Aim 2 examines the antiviral requirement for ZMP in vivo using an IAV model in zmp-/- mice. Model systems to investigate the role of ZMP deficiency in human cells are proposed. Aim 3 investigates the molecular mechanisms underlying ZMP blockade of viral entry. The role of ZMP in membrane hemifusion will be characterized. Additional studies examine ZMP dimerization and use AP-MS to identify ZMP interacting molecules which regulate antiviral function. In summary, this project will characterize the IFITM-ZMP antiviral signaling cascade and provide an improved understanding of innate antiviral defense.